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OBJECTIVES The purpose of the present study was to examine the influence of hyperbaric oxygen (HBO) on the function of osteoblastic MC3T3-E1 cells. DESIGN Murine MC3T3-E1 cells were exposed to HBO treatment (at 2.5 absolute atmospheric pressure with 100% oxygen, 90 min per day) for 28 days. Alkaline phosphatase (ALP) staining, activity, and calcium (Ca) content were measured. Gene expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1α (HIF-1α), type 1 collagen (COL1), and osteocalcin (OCN) was assessed using real-time quantitative polymerase chain reaction after a single HBO exposure for 1.5, 6, and 12 h. Furthermore, adenosine triphosphate (ATP) levels were measured using a luminescent cell viability assay. RESULTS ALP activity and Ca content were higher in the HBO group compared to those in the control group. Gene expression of bFGF, COL1, and OCN was upregulated in the HBO group; however, that of VEGF and HIF-1α significantly decreased in the HBO group in comparison with that in the control group. ATP levels were significantly higher in the HBO group compared to those in the control group. CONCLUSIONS These findings suggest that HBO accelerates bone formation by increasing the ATP levels of osteoblasts, and bFGF can act as a substitute for VEGF in vascularization by HBO application.